Lactose intolerance

Antonio Cao

Dipartimento di Scienze Biomediche e Biotecnologie

Università di Cagliari

ASL Nº8

Via Jenner, 09121 Cagliari

Mauro Congia

1) Dati Personali Data di nascita:16, Giugno 1957 Luogo di nascita:Cagliari

2) Attuale IndirizzoDipartimento di Scienze Biomediche e Biotecnologie (DSBBT), Università di Cagliari Via Jenner, 09121, Cagliari, ItalyPhone: +39-070-609 5522: Fax: +39-70-609-5558

3) Corso di studi-Laurea in medicina e chirurgia nel 1982 con 110 e dichiarazione di lode.-Specializzazione in Pediatria nel 1986 con 60/60 e dichiarazione di Lode.-Scuola di Sanità Militare a Firenze nel Gennaio-Aprile 1985 e Ufficiale Medico di Complemento Aprile 1985-Aprile 1986.

Curriculum Vitae Mauro Congia, M.D.

Obbiettivi della presentazione•Inquadramento nosologico delle varie condizioni associate alla ipolattasia

•Sintomatologia della intolleranza al lattosio, fattori che ne condizionano la variabilità e suggerimenti volti a ridurne la comparsa•Meccanismo di controllo molecolare del promotore e ipotetico meccanismo di influenza sulla trascrizione dell’mRNA del gene della lattasi che potrebbe essere alla base della ipolattasia•Genetica della lattasi, implicazioni evoluzionistiche e prospettive future

Lactose intoleranceTerminological differences and possible confounding terms about lactose intolerance

•Hypolactasia (lactase nonpersistence, lactase restriction): means that there is low lactase activity in the jejunal mucosa

•Normolactasia (lactase persistence): means that there is persistent lactase activity comparable to the neonatal period

•Lactose maldigestion and lactose malabsorption are terms to describe a poor lactose hydrolysing capacity without symptoms

•Lactose intolerance should only be used for a clinical entity, describing symptomatic lactose maldigestion (20% of hypolactasic individuals)

Lactase domainPhlorizin

hydrolase domain

Hypolactasia

•17 Exons (49 Kb)•6 Kb mRNA•1927 Amino acids

•The gene is located in 2q21

HypolactasiaIt is due to low levels of lactase-phlorizin hydrolase (LPH) in the brush border of the small-intestinal enterocytes.

Lactase has two activities:

-glucosidase activity responsible for hydrolyzing phlorizin, a disaccharide found in roots and bark of plants of the family Rosaceae and some seaweeds

-galactosidase activity -1,4- galactosidic linkage

Lactose Galactose Glucose

Lactase

Physiological factors involved in lactose absorption

•LACTOSE DIGESTIONIt is the rate-limiting step in the overall process of absorption

•Lactose is hydrolyzed

•Uptake of glucose and galactose is accomplished by the sodium-dependent glucose carrier

•Defects in this transporter result in severe diarrhea following carbohydrate intake

Hypolactasia

Enzyme activity is greatest in the mid- jejunum, decreasing both proximally and distally, resulting in minimal activity in the proximal duodenum and the terminal ileum

Lactase activity is high during infancy, when milk is the main nutrient and in the vast majority of humans decline with aging

The switch is genetically determined despite a continued intake of lactose

Hypolactasia

Lactase deficiency may be primary or secondary

•Congenital lactase deficiency (Rare)

•Developmental lactase deficiency (Prematurity)

•Adult lactase deficiency (Very common)

Three forms with primary deficiency of lactase are recognized:

Hypolactasia

A milk-free diet leads to rapid recovery, and after 6 months of age a normal diet (with milk) is well tolerated.

A forth condition of lactose intolerance having serious consequences in infants but associated with a normal presence of lactase in enterocytes is the:

•Congenital lactose intoleranceAbnormal absorption of lactose and other disaccharides from the gastric mucosa (lactosuria disappears when lactose is given intraduodenally).

Vomiting, failure to thrive, dehydration, renal tubular acidosis, aminoaciduria, liver damage, lactosuria and cataracts.

Hypolactasia

No mutation in the lactase gene (LCT) was found in a Finnish patient with congenital lactase deficiency.

•Congenital lactase deficiency

Usually, the mother notes a watery diarrhea, generally after the first feed of breast milk or at the latest within the next 10 days.

It is a rare recessive disease

Characterized by an almost total lack of LPH activity in jejunal biopsy.

Since 1966, 42 patients have been diagnosed in Finland and 18 elsewhere.

Hypolactasia

It results from low lactase levels and is a consequence of prematurity

•Developmental lactase deficiency

Lactase activity in the fetus increases late in gestation:

23rd week 10% of full-term

between 25rd and 34rd is 30% of full-term

between 34rd and 35rd 70% of full-term

Premature infants born at 28 to 32 weeks of gestation have reduced lactase activity

Hypolactasia

The prevalence of primary adult lactase deficiency varies according to race

Of the world’s population, 75% is estimated to be lactase-deficient

Race: Persons of all races are affected, with higher prevalence among Asian, African, and South American persons.

Sex: Males and females are affected equally.

Hypolactasia

Age of presentation: variable (1-2 years among Thai population to 10–20 years among Finns)

•Adult lactase deficiencyIt is the most common form of disaccharidase deficiency

Abdominal pain (crampy, often localized to the periumbilical area or lower quadrant)

Bloating

Flatulence

Borborygmi may be audible to the patient and on physical examination

Diarrhea (stools are usually bulky, frothy, and watery)

Vomiting (particularly in adolescents)

•Symptoms after the ingestion of lactose

•Variability of symptomsThere is a high difference between patients in the perception of symptoms

Hypolactasia

Lactose intolerance

•Development of symptoms of lactose intolerance is related to several factors

1. Amount of lactose in the diet

•If gastric emptying is symptoms

•Skim milk is more associated with symptoms

•Diarrhea following subtotal gastrectomy is often a result of lactose intolerance (gastric emptying is accelerated in patients with a gastrojejunostomy)

•More rapid small-intestinal transit makes symptoms more likely

2. Rate of gastric emptying

3. Small-intestinal transit time.

Hypolactasia

•Lab Studies:

Lactose tolerance test

sensitivity of 75%

specificity of 96%

The diagnosis is confirmed if the serum glucose level fails to increase by 20 g/dL above baseline

False-negative results in presence of diabetes and small bowel bacterial overgrowth

Abnormal gastrointestinal emptying can also affect the results

Hypolactasia

Measures serial blood glucose levels after oral lactose load

Administer 500 mL of milk and measure the blood glucose level

An increase of less than 9 mg/dL indicates lactose malabsorption

Milk tolerance test

Resolution of symptoms with elimination of lactose-containing food products and resumption of symptoms with the reintroduction are findings suggestive of lactose intolerance.

Dietary elimination

Hypolactasia•Lab Studies:

Thus dietary elimination should be used only for diagnosis of lactose intolerance

This is the diagnostic test of choice •Breath hydrogen test

Hypolactasia

A rise in breath hydrogen concentration greater than 20 parts per million over the baseline after lactose ingestion suggests lactase deficiency

Subjects are administered lactose, after which expired air samples are collected for 3 hours to assess hydrogen gas concentrations

This is the criterion standard

It is invasive and rarely performed

Provides definitive information

The biopsy results may be normal if deficiency is focal or patchy

•Genetic tests

They will be available soon. May be based on the detection of C/T polymorphism at position -13910 upstream the LPH gene

Hypolactasia•Small bowel biopsy

•Secondary lactase deficiency

Treatment is directed at the underlying cause

•Morbidity

Osteopenia in lactase deficiency seems to be a risk factor for osteoporosis (due to avoidance of dairy products or interference of undigested lactose with calcium absorption)

It is low and the condition is not lethal

•Possible complications

Consumption of milk in subjects with lactase persistence has been associated with an increased risk of cataract and of ovarian cancer (galactose may be implicated)

Hypolactasia

Strategies that allow lactose maldigesters to successfully incorporate dairy foods into their diets1. Consume small amounts of lactose-containing foods2. Chronic/repeated intake of lactose-containing foods allows colonic bacteria to adapt and more efficiently metabolize lactose

3. Co-ingest lactose-containing foods with a meal

4. Consider the form of the lactose-containing food Hard cheeses, chocolate and higher fat milks, and ice cream are well tolerated

5. Eat live culture yogurt

6. Utilize commercially available lactose digestive aids

•Genetics: ARI

II

III

IV

V

I

II

III

IV

V

I

II

III

IV

D2S114 LPH D2S442 D2S314 D2S2385

2

1

1

1

Chr 2

Sequence and mapping of LPH in 1988 (Mantei et al., Kruse et al.)

•GeneticsHypolactasia

Fine mapping of LPH in 1993 (Harvey et al.)

•Genetics and molecular defect transcriptional control

Hypolactasia

promoter DNA

RNA

LPH gene

•All mammals lactase genes examined so far contain binding sites for transcription factors Cdx2, HNF-1, and GATA in their promoter

•A physical association between members of GATA-4 or GATA-5 and HNF-1 results in the cooperative activation of the promoter of LPH

•The interactionoccurs through the C-terminal zinc finger and basic regions of GATA-5 and the homeodomain of HNF-1

•Genetics: cooperative activation of the LPH promoter

promoterTATAA

G

G GH

TATAA

•An analysis of Finnish families identified a single base polymorphism 13.9 kb upstream of the human lactase gene that correlates with biochemically assayed lactase non-persistence and restricted the locus to a 47-kb interval on 2q21

•GeneticsHypolactasia

Two variants -13910 and -22018 kb upstream of the lactase gene, initially found in Finnish lactose intolerance families

-13910 was also found in all French, U.S., Italian, Korean and German and African cases

•The presence of the same DNA variants in non-persistence alleles in different, distantly related populations together suggests that the persistence variant is old, occurring long before the differentiation of these populations

Hypolactasia

•the polymorphism could modify a transcription factor binding site (AP2), but the functional significance remains to be defined.

•Genetics

C/C(-13910)

T/T(-13910)

CCCCAGGC AP2 consensus sequence?

Hypolactasia•Genetics: long-range cis-acting

regulatory element

AP2? ??

CCCCAGGC

C/T 13910 G/A 22018

Distribution of lactase phenotypes

Hypolactasia

2) ”milk dependence hypothesis”:

•pastoralists in highly arid environments maintain balance of water and electrolytes through plentiful milk supply

•lactase persistence is high among all nomadic populations of sub-Saharan area (Beja in Sudan, Tuaregs in Niger, Fulani in Nigeria, Tussi in Congo basin)

•lactase persistence is high among Northern European populations. Indeed, rickets and osteomalacia were potent selective factors in the conditions of low solar irradiation characteristic of Northwestern Europe

1) "calcium dependence hypothesis”

How to explain the high prevalence of lactase persistence and its geographic distribution?

In 1997 Holden and Mace, using a comparative method that takes the problem of phylogenetic confounding into account suggested that lactase persistence distribution is consequence of adaptation to dairying.

All three hypotheses are supported by the geographic distribution of high lactose digestion capacity in adults, but are also confounded by the shared ancestry of the population.

The analysis does not support the hypothesis that lactose digestion capacity is additionally selected for either at high latitudes or in highly arid environments

Finally using maximum likelihood methods they suggested that the evolution of milking preceded the evolution of high lactose digestion.

•Genetics geographic distribution

•Thus lactase persistence is an example of Gene-Culture co-evolution

Few hours after administration, there is widespread and intense gene expression. Enzyme activity persists up to six months after a single application.In a rat model, peroral application of adeno-associated virus encoding -galactosidase (to replace missing lactase) can reverse lactose intolerance.

Future prospective : oral gene therapy

ControlAAVlac after 3 days

AAVlac after 6 months

Hypolactasia

Future prospective: Transgenic animals producing low-lactose milk•Transgenic animals carrying a hybrid gene in which the rat cDNA for LPH was placed downstream a murine mammary -specific promoter

Hypolactasia

•Female mice transgenic for this new gene not only produced lactase in the milk-producing cells, but also secreted it into the milk itself. Milk collected immediately on secretion had 50% less lactose than normal but if it was allowed to collect in the mammary gland, the level dropped even further to around 85% less lactose than usual

•ConclusionsHypolactasia

•Hypolactasia is the normal condition while persistence represents the variant

•There is no clear evidence for either conditions to predispose to other diseases

•People with hypolactasia generally can tolerate about 250 ml of milk without complains

•There are many commercially available milks and dairy products containing low amounts of lactose

•Lactase enzyme may be added to food containing lactose to increase digestibility of dairy products•A DNA test for for the -13910 polymorphism associated with hypolactasia will be soon available

Riferimenti Bibliografici1.Jarvela I., Sabri Enattah N., Kokkonen J., Varilo T., Savilahti E. & Peltonen L. (1998) Assignment of the locus for congenital lactase deficiency to 2q21, in the vicinity of but separate from the lactase-phlorizin hydrolase gene. Am J Hum Genet, 63, 1078.2.Poggi V., Sebastio, G. (1991) Molecular analysis of the lactase gene in the congenital lactase deficiency. Am. J. Hum. Genet, 49 (suppl.), 105.3.Swallow D.M., Poulter M. & Hollox E.J. (2001) Intolerance to lactose and other dietary sugars. Drug Metab Dispos, 29, 513.4.Enattah N.S., Sahi T., Savilahti E., Terwilliger J.D., Peltonen L. & Jarvela I. (2002) Identification of a variant associated with adult-type hypolactasia. Nat Genet, 30, 233.